Viral hemorrhagic fevers caused by viruses belonging to the genus Ebolavirus and Marburgvirus, both members of the Filoviridae family, are known to be among the most severe infectious diseases in human and nonhuman primates (NHPs), and no licensed vaccines or effective therapeutics are currently available. Ebola virus (EBOV), in particular, has been responsible for multiple Ebola hemorrhagic fever (EHF) outbreaks with case-fatality rates ranging from 65 to 90%. Studies with animal models and limited clinical data from EHF outbreaks suggest that interdependent pathogenic processes, including both the host immune and pathophysiological responses, induced by EBOV infection trigger severe hemorrhagic syndrome. However, in order to develop effective treatments for EHF, it is necessary to better understand the mechanisms of viral and host interactions at the molecular and cellular levels and how these interactions contribute to the in vivo pathogenic process. Therefore, our research is focused on elucidating the roles of viral proteins in the viral replication cycle and pathogenesis. To accomplish this, we have four ongoing projects: (1) the characterization of pathogenic processes in the Syrian hamster model that recapitulates EHF, (2) the development of efficient reverse genetics systems for generating recombinant EBOV from cDNA, (3) the elucidation of the molecular mechanisms of EBOV adaptation in mice, and (4) the characterization of EBOV viral protein interactions. (1) The characterization of pathogenic processes in the Syrian hamster model, which recapitulates EHF. While the NHP model is used to evaluate the efficacy of EBOV vaccines and therapeutics because it accurately recapitulates disease, rodent models (mice and guinea pigs) are convenient and suitable for elucidating the roles of specific viral proteins in the pathogenic process and have been widely used in numerous aspects of EBOV research. However, rodent models produce only limited and inconsistent coagulation abnormalities, which are a hallmark clinical feature of EHF. Therefore, we have developed and characterized a novel lethal Syrian hamster model of EHF based on infection with mouse-adapted (MA)-EBOV that manifests many of the clinical and pathological findings observed in EBOV-infected NHPs and humans, including coagulation abnormalities. To determine the mechanisms of pathogenesis in this model, we characterized and compared host responses induced as a result of lethal (MA-EBOV) versus non-lethal infection (WT-EBOV). Our comprehensive virological, pathological, physiological, and immunological analyses revealed that suppression of type I interferon responses and induction of coagulation abnormalities by MA-EBOV are, along with the uncontrolled pro-inflammatory response, key events contributing to the ultimate death of the animals. Our novel hamster model will facilitate research on pathogenesis and countermeasure development. (2) The development of efficient reverse genetics systems for generating recombinant EBOV from cDNA. We have been developing a more efficient rescue system to generate recombinant EBOV. Full-length wild-type and mouse-adapted EBOV genome clones have been constructed with the hammerhead ribozyme sequence inserted at the 5 terminus, resulting in the production of an authentic 5 terminus in the subsequent viral cRNA. Using this system, we have significantly improved the rescue efficiency of recombinant EBOV over the previous system lacking the hammerhead ribozyme. Additionally, by comparing the rescue efficiencies of this system in a variety of different cell lines, we have determined that Huh7 cells produce significantly higher titers of virus than any other cell line tested, including Vero cells, which have been traditionally used in EBOV rescue systems. In fact, we found that the genomes of EBOV rescued in Vero cells were unstable, accruing significantly more nucleotide substitutions and insertions than the genomes of viruses rescued in Huh7 cells. Overall, we have succeeded in developing an EBOV reverse genetics system that is significantly improvedin both efficiency and fidelityover previous systems. Not only has this system improved our ability to rescue recombinant wild-type and mouse-adapted EBOV, but we anticipate that it will also improve our ability to rescue and characterize mutant viruses that may have depressed growth characteristics. (3) The elucidation of the molecular mechanisms of EBOV adaptation in mice. While adult immunocompetent mice resist EBOV infection, MA-EBOV causes lethal infection in mice. Previously, a study using EBOV reverse genetics identified that amino acid substitutions in VP24 and nucleoprotein (NP) were primarily responsible for the acquisition of virulence in mice. In order to uncover the molecular mechanisms underlying EBOV pathogenesis in the mouse model, we have generated a series of recombinant viruses possessing various combinations of wild-type and mouse-adapted genes, with particular focus on mutations in the NP and VP24 genes. Growth comparison of these mutants in mouse cell lines suggested that mutations in NP and VP24 enhanced the growth ability of EBOV in target cells. We will examine the ability of the mutations in NP and VP24 to enhance viral replication and modify the host response in target cells at the early phase of infection in mice. (4) The characterization of EBOV viral protein interactions. Relatively little information exists regarding the molecular details that govern interactions between EBOV proteins. As such, we are actively interested in understanding the determinants of EBOV protein interaction and the functional outcomes of those interactions. For example, it is thought that NP, the EBOV nucleoprotein, relies in part on an interaction with VP24, the so-called minor matrix protein, for the efficient production of nucleocapsids. Moreover, we have previously demonstrated that two point mutations, one each in NP and VP24, are necessary and sufficient for conferring pathogenicity to EBOV in mice, perhaps suggesting a further functional relationship between these two proteins. Nevertheless, the molecular basis for this interaction is not known. To address this, we have raised a series of rabbit and guinea-pig polyclonal antibodies against several VP24 peptides and demonstrated that these antibodies recognize VP24 by immunoblot, immunoprecipitation, and immunofluorescence. Considering that no commercial antibody exists for detecting VP24 and that epitope-tagging this protein is difficult, possessing an antibody that robustly recognizes VP24 is key to all further downstream experiments. Accordingly, we have used this antibody in co-immunoprecipitation assays to repeat and confirm the interaction between NP and VP24, and we are currently in the process of mapping the regions of NP and VP24 that are critical for this interaction. To this end, we have produced a 3D model of EBOV VP24 based on the recently published structures of Sudan and Reston VP24. This model, and future in silico work with NP, will aid in elucidating the molecular determinants of the NP-VP24 interaction.